Project Summary Drug addiction is a debilitating disorder that affects ~25 million Americans?ages 12 and up?with treatment costs averaging >$190 billion annually in the United States alone. Drug abuse is characterized by loss of control over drug intake, as well as persistent drug-seeking behaviors, despite negative consequences to both the drug abuser and those directly affected by their behavior. Given that drug addicts continue to crave and pursue drugs of abuse following extended periods of abstinence and/or treatment indicates that there must be life-long changes that occur in brain to promote these behavioral phenotypes. Persistent changes in neuronal gene expression are known to promote physiological alterations implicated in drug addiction. More recently, cell-type and brain region specific epigenetic mechanisms have also been demonstrated to regulate transcriptional programs contributing to addiction-like behaviors; however, our understanding of how these mechanisms mediate life-long addiction remains limited. Dopaminergic neurotransmission in the central nervous system plays a critical role in psychostimulant-induced neural plasticity, with alterations in dopamine production/function being implicated in both the development and treatment of substance abuse disorders. Although packaging of dopamine by the vesicular monoamine transporter is essential for numerous aspects of reward, recent data have demonstrated the additional presence of `reserve' pools of extravesicular monoamines in the nucleus of monoamine producing neurons. Dopamine, as well as other monoamines, have previously been shown to form covalent bonds with certain cytoplasmic proteins catalyzed by the tissue Transglutaminase 2 enzyme. Dr. Maze's laboratory has recently identified histone proteins as robust substrates for dopaminylation in vivo, specifically on histone 3 glutamine 5 (H3Q5dop). Furthermore, our preliminary data, collected under the co-mentorship of Dr. Kenny, demonstrate that chronic withdrawal from volitional administration of extended access to cocaine in rodents results in high levels of dopamine accumulation in the nucleus of dopamine producing neurons in the ventral tegmental area (VTA), as well as altered expression of Tgm2, the H3 dopaminylase. In this proposal, I will therefore investigate potential roles for H3Q5dop in cocaine-induced transcriptional plasticity in VTA via a combination of next generation sequencing approaches (Aim 1). Next, I will utilize novel viral-mediated techniques to overexpress or inhibit H3Q5dopaminylation in this region to directly investigate functions for histone dopaminylation in cocaine- seeking behaviors (Aim 2). Taken together, these potentially paradigm-shifting studies will aid in our understanding as to how monoamines, specifically dopamine, function in brain to regulate neurotransmission independent neuronal plasticity and cocaine-mediated behaviors. !